MIR-122 and DDX6 facilitate hepatitis C virus replication via independent mechanisms



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Globally 130 million people are infected with hepatitis C virus (HCV) that leads to chronic hepatitis. Current therapy is limited in its efficacy; hence better interventions are required. A detailed understanding of the host factors involved in HCV replication such as miR-122 and DEAD-box RNA helicase, DDX6 may facilitate development of novel therapies. miR-122 is a liver-specific microRNA that is required for efficient replication of HCV in hepatocytes. Mutational analyses of the genotype 1a/2a chimeric HCV (HJ3-5) genomes indicated that miR-122 promotes replication by binding directly to the two miR-122 binding sites in the genomic RNA and, at least in part, by stimulating internal ribosome entry site-mediated translation. A comparison of mutants with substitutions in only one site revealed S1 to be dominant over S2 site for its role in viral replication. However, a comparison of the replication capacities of the double binding-site mutant and an IRES mutant with a quantitatively equivalent defect in translation suggests that the decrement in translation associated with loss of miR-122 binding is insufficient to explain the profound defect in virus production by the double mutant. miR-122 is thus likely to act at an additional step in the virus lifecycle. Overexpression and knockdown experiments indicated that DDX6, a miRISC component that is upregulated in liver during chronic hepatitis C, is another cellular factor supporting HCV replication. DDX6 forms a complex containing HCV core protein, and both viral and cellular RNAs via its helicase domain and its helicase activity is required to facilitate virus replication. However, the relevance of DDX6-core interaction is unclear as DDX6 abundance influenced the replication of subgenomic replicon RNAs lacking core sequence. Importantly, DDX6 knockdown caused minimal reductions in cellular proliferation, generally stimulated cellular translation ([35S]-Met incorporation), and did not impair translation directed by the HCV internal ribosome entry site. Thus, DDX6 helicase activity is essential for efficient HCV replication, reflecting essential roles for DDX6 in HCV genome amplification and/or maintenance of cellular homeostasis. Finally, DDX6 knockdown did not impair miR-122 biogenesis nor alter HCV responsiveness to miR-122 supplementation suggesting that DDX6 and miR-122 facilitate HCV replication via independent mechanisms.